Decoding molecular plasticity of the dark proteome

Abstract

The mechanisms by which intrinsically disordered proteins (IDPs) engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function. Nuclear transport receptors (NTRs) can move through the central channel of the NPC which is filled with hundreds of phenylalanine‐glycine‐rich nucleoporins (FG‐Nups) reaching millimolar concentrations with elusive conformational plasticity. Since site‐specific labeling of proteins with small but highly photostable fluorescent dyes inside cells remains the major bottleneck for directly studying protein dynamics in the cellular interior, we have now developed a semi‐synthetic strategy based on novel artificial amino acids that are easily and site‐specifically introduced into any protein by the natural machinery of the living cell via a newly developed thin‐film synthetic organelle that equips the living cell with up to three genetic codes. This allowed us to develop an experimental approach combining site‐specific fluorescent labeling of IDPs in non‐fixed cells with fluorescent lifetime imaging microscopy (FLIM) to directly decipher the plasticity of FG‐Nups via FRET. Our study enabled a conformational look on the condensated IDPs in the sub‐resolution (roughly (50 nm)3 small cavity) cavity of the NPC. By measuring the end‐to‐end distances of different segments of the labeled FG‐Nups using FLIM‐FRET, we can extract the scaling exponent, which directly describes the conformations of FG‐Nups at their functional status as well as the solvent quality in the cellular and even inner NPC environment.Reinkemeier CD, Lemke EA. Dual film‐like organelles enable spatial separation of orthogonal eukaryotic translation. Cell. 2021 Sep 16;184(19):4886‐4903.e21Celetti G, Paci G, Caria J, VanDelinder V, Bachand G, Lemke EA. The liquid state of FG‐nucleoporins mimics permeability barrier properties of nuclear pore complexes. J Cell Bio. (2020) Jan 6;219(1).Reinkemeier CD, Estrada Girona G, Lemke EA, 2019 Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. Science, Mar 29;363(6434)Nikić I, Estrada Girona G, Kang JH, Paci G, rei S, Koehler C, Shymanska NV, Ventura Santos C, Spitz D, Lemke EA, Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy. Angew Chem Int Ed Engl. (2016) Dec 23;55(52):16172‐16176